In recent years, considerable evidence has amassed for the idea that proteases, by cleaving the α or γ subunit of ENaC, can increase the activity of this channel (Hamm et al, 2010). This evidence has come from in vitro studies, largely using electrophysiological techniques to assess ENaC activity in heterologous expression systems or in isolated nephron segments (Kleyman et al, 2009; Nesterov et al, 2008). The present study has assessed the physiological relevance of these observations by measuring sodium reabsorption in rat collecting ducts in vivo. Rats (n = 14) on a standard sodium intake were anaesthetised with thiobutabarbital sodium (Sigma; 110 mg kg-1, I.P.) and prepared surgically for micropuncture. Late distal tubules of superficial nephrons were perfused twice (5 nl min-1 for 3-6 min) with a solution similar to native tubular fluid but containing 14C-inulin and 22 Na. The first perfusion was always a control solution; the second perfusion was either a control solution (time controls) or a solution containing chymotrypsin at a concentration of 2 µg ml-1. Urinary recoveries of 14C-inulin and 22Na were monitored; if the inulin recovery was >85% of the amount perfused, the urinary 22Na/14C-inulin (Na/In) ratio was calculated. In time controls, the Na/In ratio did not change significantly (34.3 ± 3.0% vs. 37.0 ± 2.9%; n = 18 pairs, NS, paired t test). In contrast, chymotrypsin reduced the ratio from 34.1 ± 4.0% to 25.6 ± 2.9% (n = 14 pairs, P<0.05, paired t test). These data are consistent with the proposal that proteases, acting from within the lumen, can stimulate ENaC activity in the mammalian collecting duct.